NZ562149A - Cultures of E1-immortalized cells and processes for culturing the same to increase product yields therefrom - Google Patents

Abstract

Disclosed is a culture of cells derived from PER.C6 cells, characterized in that said culture comprises at least 12 x 106 cells/ml.

Description

*10054692177* 56 2 149 \ -1 OCT 2007 | \rECETV^ NEW ZEALAND PATENTS ACT, 1953 No: Date: Divided out of New Zealand Specification No. 542585 dated 6 May 2004 COMPLETE SPECIFICATION CULTURES OF El-IMMORTALIZED CELLS AND PROCESSES FOR CULTURING THE SAME TO INCREASE PRODUCT YIELDS THEREFROM We, CRUCELL HOLLAND B.V., Archimedesweg 4, NL-2333 CN Leiden, The Netherlands, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page 1 a) la TITLE OF THE INVENTION Cultures of El-immortalized cells and processes for culturing the same to increase product yields therefrom.

This is a divisional of New Zealand Specification No. 542585.

FIELD OF THE INVENTION The invention relates to the field of cell culture. In particular, the invention relates to the field of culturing cells derived from cells that have been immortalized with El sequences from adenovirus. More in particular, the invention 10 relates to culturing such cells to obtain high levels of products from such cells.

BACKGROUND OF THE INVENTION A human PER.C6® cell line, exemplified by cells deposited at the ECACC under no. 96022940, derived from retina cells by immortalisation with the adenovirus (Ad5) Ela and Elb genes is disclosed in US patent 5,994,128. Besides the ability to function as packaging cells for El-deleted adenoviral vectors (US patent 5,994,128; WO 01/005945), and for producing other viruses (WO 01/38362), El-immortalized cells, such as PER.C6 cells, can be used to produce recombinant proteins, such as antibodies (WO 00/63403).

Xie et al (2002) have disclosed a process for serum-free suspension cultivation of El-immortalized cells. However, the product yields obtained using the culturing processes disclosed in the art for El-immortalized cells, can be improved. It is an object of the present invention to provide novel processes to increase the product yield from this type of cells; and/or to provide the public with a useful choice.

BRIEF DESCRIPTION OF THE FIGURES intellectual property office of n z - 2 APR MB received 3 Table 2) for clone 1. A: viable cell numbers (Nv) . B: antibody (Ab) concentration. Circles: batch. Squares: fed-batch.

Arrows: last feed.

Fig. 8. Result of further improved modified feed with different first and subsequent feed additions (example 4, Table 2) for clone 2. Nv: viable cell number. Ab: antibody concentration.

Fig. 9. Galactosylation levels of IgG produced according to processes according to invention.

Fig. 10. Result of further improved modified feed with different first and subsequent feed additions (example 4, Table 2) for clone 3. Nv: viable cell numbers. Ab: antibody concentration.

SUMMARY OF THE INVENTION In one aspect the invention provides a culture of cells derived from PER.C6 cells, characterized in 6 that said culture comprises at least 12 x 10 cells/ml.

Parent specification no. 542585 is directed to feed strategies for fed-invention of NZ 542585 provides a method for the culturing of such cells, said cells capable of growing in suspension, comprising the 25 steps of: determining at least once during the culturing of the cells the concentration of at least one medium component selected from the group consisting of glucose, glutamine phosphate, leucine, serine, isoleucine, arginine, methionine, cystine, valine, lysine, threonine and glycine, adding 30 components to the medium during the culturing of the cells at or prior to the depletion of at least one of the components of which the concentration was determined in the previous step, - 2 APR 20® rfceived 4 wherein the components added at least comprise glucose, glutamine, phosphate, leucine, serine, isoleucine, arginine, methionine and cystine. Other components that beneficially may be added to the invention of NZ 542585, amounts and time of 5 addition of the components are provided herein below.

It is another aspect of the invention of NZ 542585 to provide a culture of cells produced by the methods of the invention.

Also described is a culture of cells derived from cells immortalized by adenovirus El sequences, characterized in that said culture comprises at ' least 10 x 106 cells/ml. Preferably, said culture comprises at least 12 x 106 cells/ml, more preferably at least 15 x 10s cells/ml. In certain preferred embodiments the culture according to the invention comprises more than 20 x 106, 25 x 106, 30 x 106 or 40 x 106 cells/ml. Methods to obtain such cultures are also provided herein.

Also described is a method to increase cell densities and product yields from a culture of cells immortalized by adenovirus El sequences is provided. In one embodiment hereof, a process for culturing such cells is provided, characterized in that said process comprises a step of subculturing said cells at a seeding concentration of between 0.8 x 106 and 2.0 x 106 viable cells/ml, preferably between 0.9 x 106 and 1.5 x 106 viable cells/ml.

Also described is a method for producing a product in cells derived from PER.C6 cells, said cells being in a culture medium, wherein said product is chosen from the group consisting of a recombinant protein, a virus, and a recombinant adenovirus with a deletion in the El region, characterized in that the culture medium is supplemented by addition of at least Glutamine, Glucose, Phosphate, Leucine, Serine, Isoleucine, Arginine, Methionine and Cystine to the culture medium.

Also described is a method for producing a product in cells derived from PER.C6 cells, wherein said cells are cultured in a culture medium, characterized in that the culture medium is supplemented by adding the following components to the culture medium per liter: 3.6-21.6 mmoles glucose, 6.8-40.9 mmoles glutamine, 0.40-2.4 mmoles leucine, 2.31-13.9 mmoles serine, 0.3-1.8 mmoles isoleucine, 0.28-1.66 mmoles arginine, 0.14-0.83 mmoles methionine, 0.15-0.9 mmoles cystine, 0.27-1.62 mmoles valine, 0.26-1.58 mmoles lysine, 0.18-1.08 mmoles threonine, 0.06-0.36 mmoles asparagine, 0.078-0.47 mmoles tyrosine, 0.06-0.36 mmoles histidine, 0.012-0.072 mmoles phenylalanine, 0.036-0.22 mmoles tryptophan and 0.45-2.7 mmoles phosphate.

The cells used in the methods described herein are de Other cells can also be used, such as retina cells, more pr embryonic retina (HER) cells, such the ECACC no. 96022940.

In certain embodiments, said cells can produce recombinant proteins, preferably antibodies, at high yields. In other embodiments said cells comprise recombinant adenoviral vectors having a deletion in.the El-region, or other viruses, which INTELLECTUAL property OFFICE of ins z ■iwdfro^^C^'s. jferably from human fiii QpEbitVi EEnDc 1I52199_1.DOC QfBQE OF Hi -2 APR 2M received can be produced on said cells in high yields using the process according to the invention. In preferred embodiments, the cells are cultured at least part of the time in a serum-free medium.

By "comprising" is meant 'consisting at least in part of, that is to say when interpreting independent claims including that term, the features prefaced by that term in each claim all need to be present but other features can also be present.

DETAILED DESCRIPTION OF THE INVENTION The productivity of any cell line is mainly defined by three basic parameters, the specific productivity of the cell line, the peak viable cell concentration that is attainable and the 10 length of the production process that is possible. Increases in either of these variables will lead to increases in the final product concentration and is dependent to a large extent on the cell line. In a straight batch culture, cell lines such as CHO and SP2/0 can achieve cell densities up to 4 x 106/ml. 15 In fed-batch or perfusion processes the viable cell concentration is increased, and typically hybridoma cells such as SP2/0 can be cultured up to 10 x 106 cells/ml, while CHO can be cultured up to 6-10 x 106 cells/ml. The invention describes methods to increase the viable cell density of cultures of 20 cells derived from PER.C6 cells, to attain cell densities beyond those reported in the prior art. Furthermore, the methods described herein can be used to obtain higher product yields from cultures of cells according to the invention.

The present invention provides a culture of cells derived from PER.C6 cells, characterized in that said culture comprises at least 12 x 106 cells/ml.

Described herein are improvements on how El-immortalized cells, i.e., PER.C6 cells, can advantageously 30 be used for the production of high yields of monoclonal antibodies. It is disclosed that these cells be cultured to very high viable cell concentrations in a straight batch process (up to 14 x 106 viable cells/ml). - 2 APR R F. c eiv e p 6 Furthermore, PER.C6 cells, are well suited to a fed-batch process as a culture of these cells unexpectedly consumes lactate and ammonia and maintains viability for long periods of time under nutrient limiting S conditions. Methods to increase product yields from said cells by a feed strategy in cultures are provided herein.

With the term nfeed strategy' as used herein is meant the addition of certain identified components including but not limited to nutrients, such as sugars, amino acids, and the 10 like, to the culture medium. The identified components are preferably added in certain amounts and at certain times, when they aire required to improve product yields from the cells, such as provided herein.

The El-immortalized cells, PER.C6 cells, are also 15 well suited to a perfusion process as they can be maintained at very high viable cell concentrations (up to 50 x 106 cells/ml with a viability of at least 85%) for long periods of time and with good final product concentrations.

Culture media The processes described herein generally increase the product yields from the cells compared to yields obtained with processes described in the art for the cells according to the invention. Preferably, serum-free culture media are used at 25 least part of the time in the processes according to the invention. Preferably, the medium contains only recombinantly produced proteins, which are not of animal origin. Such culture media are commercially available from various sources. In one embodiment ■ VPRO culture medium (JRH Biosciences) is used for the fed-batch or (fed-)perfusion process. 1 Products The methods described herein are preferably used to produce products in cells of the invention. The processes described herein can be used for the improved production 5 of antibodies, as well as other proteins (WO 00/63403). For the production of proteins, the cells of the invention suitably comprise nucleic acid encoding said proteins, in operable association with elements capable of driving expression of said proteins. Furthermore, the processes can be 10 used for improvement of the production of recombinant adenoviral vectors having a deletion in the El-region, in which case the cells are used as complementing cells, which in itself is known to the skilled person according to established methodology (e.g. US patent 5,994,128; WO 01/005945). 15 Moreover, the processes described herein can be used to improve a process for propagation of other (non-adenovirus) viruses in the cells (WO 01/38362). Hence, products described herein can be recombinant proteins, such as antibodies, erythropoietin, and the like, as well as 20 recombinant adenoviral vectors with a deletion in the El region, or other viruses.

Cells The cells described herein are cells that have 25 been immortalized with El sequences from an adenovirus, which cells are also referred to herein as El-immortalized cells. Such cells express at least a functional part of the E1A region of an adenovirus, and preferably also at least a functional part of the E1B region. ElA protein has 30 transforming activity, while E1B protein has anti-apoptotic activities. The cells described herein may be derived from any cell, including lung cells, kidney cells, INTELLECTUAL OFFICE OF .N;]"rtrvi received 8 amniocytes, but preferably are derived from retina cells. They may be derived from embryonic retina cells. Preferably the cells according to the invention are human cells. A method for immortalization of embryonic retina cells has been described 5 in the art (US patent 5,994,128). Accordingly, a retina cell that has been immortalized with El sequences from adenovirus can be obtained by that method.

The cells of the invention are derived from the El- immortalized HER cells, PER.C6 cells. PER.C6 cells for the purpose of the present application shall mean cells from an upstream or downstream passage or a descendent of an upstream or downstream passage of cells as deposited under ECACC no. 96022940. In addition, also the E2A region with a tsl25 mutation may be present (see e.g. US patent 6,395,519) 15 in said cell. A cell derived from a PER.C6 cell can be a PER.C6 cell infected with recombinant adenovirus or other virus, and can also be a PER.C6 cell into which recombinant nucleic acid has been introduced, e.g. comprising an expression cassette wherein nucleic acid encoding a protein of 20 interest is operably linked to sequences capable of driving expression thereof, such as a promoter and polyA signal, wherein preferably said cells are from a stable clone that can be selected according to standard procedures known to the person skilled in the art. A culture of such a clone is 25 capable of producing a protein encoded by said recombinant nucleic acid.

Components for feed strategies Described herein are processes for 30 culturing cells according to the invention, wherein by feed strategies certain amino acids are added during the culturing process to replenish amino acids of intellectual >>. office or £ -2 m.

RCPCiw 9 which the concentration has become or will become limiting for an optimal process and product yields. By amino acid is intended all naturally occurring alpha amino acids in both their D and L stereoisomeric forms, and their derivatives. A 5 derivative is defined as an amino acid that has another molecule or atom attached to it. Derivatives would include, for example, acetylation of an amino group, amination of a carboxyl group, or oxidation of the sulfur residues of two cysteines to form cystine. Further, amino acid derivatives may 10 include esters, salts, such as chlorides, sulphates, and the like, as well•as hydrates. It will be understood by the person skilled in the art, that where a specific amino acid is mentioned herein, a derivative may also be used and is meant to be included within the scope of the disclosure. Other 15 components such as sugars, growth factors, vitamins, etc may also be added to improve the processes described herein.

Feed strategies In one aspect, described is a method for 20 producing a product in cells derived from PER.C6 cells, in a culture medium, wherein said product is chosen from the group consisting of a recombinant protein, a virus, and a recombinant adenovirus, with a deletion in the El region, characterized in that said method comprises a step wherein at 25 least Leucine, Serine, Isoleucine, Arginine, Methionine and Cystine are added to the culture medium. In one aspect described is a method for the culturing of cells derived for PER.C6 cells, said cells capable of growing in suspension, comprising the steps of: determining at 30 least once during the culturing of the cells the concentration of at least one medium component selected from the group consisting of glucose, glutamine, phosphate, leucine, serine. intellectual property office of h\Z. - 2 APR m R E ft c it/ c r> isoleucine, arginine, methionine, cystine, valine, lysine, threonine and glycine, adding components to the medium during the culturing of the cells at or prior to the depletion of at least one of the components of which the concentration was 5 determined in the previous step, wherein the components added at least comprise glucose, glutamine, phosphate, leucine, serine, isoleucine, arginine, methionine and cystine. "Depletion" as used herein is defined as the time a component has a concentration of 30% or less of the starting 10 concentration in the culture medium. In these aspects, the determination of the concentration of at least one medium component selected from the group consisting of glucose, glutamine, phosphate, leucine, serine, isoleucine, arginine, methionine or cystine is preferred over the determination of 15 only components selected from the group consisting of valine, lysine, threonine and glycine. In certain embodiments, the concentration of at least two medium components is determined in the first step. In certain embodiments, the components that are added further comprise 20 one or more of valine, lysine, threonine, glycine, asparagine, tyrosine, histidine, phenylalanine, tryptophane, calcium, LongR3 IGF-1, Long EGF and insulin. In specific embodiments, the components are added in an end concentration in mmoles/1 of freshly added component per 10 x 106 cells/ml of 6.0 for 25 glucose, 2.60 in the first feed and 1.75 in subsequent feeds for glutamine, 0.70 for phosphate, 0.66 for leucine, 1.10 in the first feed and 0.55 in subsequent feeds for serine, 0.50 for isoleucine, 0.46 for arginine, 0.23 for methionine, and 0.25 for cystine. In further embodiments, the following 30 components are further added to an end concentration in mmoles/1 of freshly added component per 10 x 1Q6 cells/ml of 0.45 for valine, 0.44 for lysine, and 0.30 for threonine. In 11 further embodiments, the following components are further added to an end concentration in mmoles/1 of freshly added component per 10 x 10s cells/ml of 0.10 for asparagine, 0.13 for tyrosine, 0.10 for histidine, 0.02 for phenylalanine, and 5 0.06 for tryptophan. Furthermore, calcium may be added in an end concentration in mmoles/1 of freshly added component per 10 x 106 cells/ml of 0.02. Growth factors such as IGF, EGF, and insulin or their derivatives may also suitable be present in the growth medium. The amounts for the addition of components 10 above may have an error margin per component of 33% or less, preferably 20% or less, more preferably 10% or less, even more preferably 5% or less. The amounts are presented per 10 x 106 cells/ml, and are linearly dependent on the number of cells/ml. In preferred embodiments, said components are added 15 at between 48 hours and the moment of depletion of at least one of the medium components the concentration of which was determined in the previous step. In certain embodiments, said addition is at a time between 24 hours and just prior to depletion. In certain aspects, described is a method 20 wherein said cells express a recombinant immunoglobulin that is secreted into the culture medium to a level of at least 500 mg per liter, preferably at least 700 mg/1, more preferably at least 850 mg/1, even more preferably at least 1000 mg/1, still more preferably at least 25 1250 mg/1, still more preferably at least 1500 mg/1, still more preferably at least 1750 mg/1 and still more preferably at least 2000 mg/1. In general, the addition of medium components described herein, ie. in for instance a fed-batch process, results in an increase in the yield of 30 produced product of at least 1.5x, preferably at least. 2x, more preferably at least 2.5x and still more preferably about ■2 APR2W9 12 3x or even higher, compared to the process wherein no components are added, i.e. the batch process.

In addition to use in a fed-batch process, the feed strategies described herein can also be beneficially used in 5 an optimized batch process, as set out in example 5. entire culture medium may be exchanged. It is shown that 10 unexpected high viable cell densities can be attained when this is applied to cells derived from PER.C6 cells medium may be performed by any means known to the person skilled in the art, including but not limited to collection of 15 the cells by centrifugation, filtration, and the like, followed by re-suspension of the cells into fresh culture medium. Alternatively, a perfusion system may be used, wherein culture medium is either continuously or intermittently exchanged using a cell separation device such as a centritech 20 centrifuge or passage through a hollow fibre cartridge, and the like. Therefore also described is cells derived from PER.C6 cells characterized in that culture medium is exchanged at a rate of 25 0.2-3, preferably 0.5-3, culture volumes per day (24 hours). Cultures obtained using this method preferably have viable cell densities higher than 20 x 106 cells/ml, more preferably higher than 30 x 10s cells/ml. In certain aspects, such cultures have cell densities higher than 40 x 106 cells/ml. In 30 certain aspects such cultures are used to produce recombinant antibodies with a yield of at least 150 mg/l/day, preferably at least 200 mg/l/day, more preferably at least 300, 400, or Perfusion A1ternatively, the Exchanging culture a process for culturing cells derived from 13 500 mg/l/day. Of course, also other products can be produced by such methods. It is shown in here that one complete volume exchange of culture medium each day supports at least 30 x 106 viable cells/ml with antibody yields 5 of more than 500 mg/L/day (up to 750 mg/l/day) (Fig. 5). One complete medium exchange per day corresponds to a continuous perfusion rate of 3 volumes per day, meaning that a continuous perfusion system could yield approximately at least 150-200 mg/L/day. One method to reduce this perfusion rate and thus 10 increase antibody yields (by reducing the volume in which the antibody is secreted) is to supplement the fresh culture medium with the essential components (knov^j as fed-perfusion). These components for antibody-producing El-immortalized cell, i.e., a.PER.C6 cells, clones are identified herein (see example 15 2). Therefore described is such a fed-perfusion system, wherein the feed strategies described herein are employed. A common drawback of fed-perfusion processes is the build-up of toxic metabolic by-products (such as lactate and ammonia), 20 which can result in low cell viabilities and product yields.

There is often a requirement at high cell concentrations for a high perfusion rate to remove these by-products. One advantage demonstrated for El-immortalized cell, i.e., a PER.C6 cell, clones according to the invention is that they are capable of 25 utilising lactate and ammonia such that concentrations do not become problematical (see Fig. 3A). It is therefore possible to obtain an antibody yield of at least 500 mg/l/day by changing the culture medium once or twice a day. Alternatively this can be achieved by using a continuous perfusion rate of 30 for instance 1 volume per day in combination with supplementation of the medium with a feed concentrate (fed- 14 perfusion). This can advantageously be combined with a cell bleed (removing a certain percentage of the cells population).

Cultures with high cell densities are advantageous for obtaining high product yields. It is therefore another aspect 5 of the invention to provide a culture of cells derived from PER. C6, cells, said culture comprising at least 12 x 106 cells/ml. The viability in the culture is at least 80%. Preferably, the viability is at least 90%, more preferably at least 95%. The cultures according to 10 the invention are preferably suspension cultures, meaning that the cells in said cultures are in suspension in the culture medium, such as in shake flasks, roller bottles, bioreactors, including stirred tanks, air lift reactors, and the like. The strategies disclosed herein may however also be used for 15 cultures of cells in hollow fiber reactors, such as described by Tanase et al (1997), and for adherent cultures, such as cells on microcarriers. In one embodiment, said culture comprises at least 15 x 106 cells/ml. It is disclosed herein that up to 14 x 106 cells/ml can be obtained by a straight 20 batch culture.

It is further demonstrated that, using medium perfusion, even higher cell densities can be achieved, up to 50 x 106 cells/ml. The prior art does not provide any indication that such unexpected high cell densities are obtainable. In other 25 preferred embodiments therefore, the invention provides a culture of cells derived PER.C6 cells, said culture comprising at least 15 x 10s cells/ml, preferably at least 20 x 106 cells/ml, more preferably at least 25 x 106 cells/ml. In 30 specific embodiments, said culture comprises at least 30 x 106 cells/ml, or even at least 40 x 106 cells/ml. Cultures with at least 15 x 106 cells/ml according to the invention appear r office 6f'iz yl ' 2 APR ece( veni obtainable by a perfusion process, meaning that culture medium is exchanged during the culturing process. The cultures according to the invention have a viability of at least 80%, preferably at least 85%, more preferably at least 90%, still 5 more preferably at least 95%. Said cultures are suspension cultures. Said cultures further comprise growth medium. Said growth medium preferably is serum-free. The cells of the culture may comprise recombinant nucleic acid molecules encoding immunoglobulins, or parts or derivatives thereof, in 10 expressible format. Such cells are capable of producing immunoglobulins in high yields. In particular, it is shown herein that a culture of cells according to the invention, wherein the medium is exchanged every day, and wherein more than 30 x 10s cells/ml are present, can provide recombinant 15 antibody yields of at least 500 mg/l/day. The cells in said culture preferably produce at least 10 pg protein/cell/day.

The processes described herein, especially those for recombinant protein production, can also be combined with 20 other measures described in the art that in some cases improve product yields. Therefore, in certain embodiments of the invention the culture medium is subjected to a temperature shift before or during the production phase, e.g. by running the process at a lower temperature, e.g. between 30°C and 25 35°C, in the production phase (see e.g. US patent 6,506,598, and literature cited therein, which describes effects of lowering the cell culture temperature on several parameters for recombinant protein production), or by the addition of cold culture medium to the culture (wherein cold is meant to 30 be lower than the temperature the cells are cultured in, preferably the cold culture medium having a temperature between 2°C and 8°C) when the cells are subcultured or later intellectual property OhFICE of t\i z. - 2 APR 2009 recei v e d 16 during the culture process. In other embodiments, specific growth factors may be added to improve the processes described herein with regard to product yields. In yet other embodiments for the production of proteins, the processes 5 described herein may be improved by the addition of alkanoic acids or salts thereof, such as sodium butyrate, either during the whole culture phase or only during the production phase (see e.g. US 6,413,746, and references therein, which describes effects of addition of butyrate on 10 production of proteins in cell culture). In yet other embodiments for the production of proteins, the culture medium is subj ected to a temperature or pH shift (Weidemann et al 1994, Sauer et al 2000).

It will be clear to the person skilled in the art that several aspects and/or embodiments according to the invention can be combined to provide a process for culturing cells which leads to particularly good product yields. As a non-limiting example, it is for instance possible to seed a culture of El-20 immortalized cells at about 0.8 x 10® to 2.0 x 106 cells/ml, and use a feed strategy and/or exchange the growth medium during the culturing process to improve the final product yields.

The invention will now be illustrated with some examples, not 25 intended to limit the scope of the invention.

Experimeatal Methods and vectors for genetically engineering cells and/or cell lines to express a protein of interest are well known to 30 - those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly intellectual property office of n2 ' I APR ffius rec 17 updates) and Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989). General and standard cell culture techniques are known to the person skilled in the art, and are for instance described in R.I.

Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9). Such standard techniques were followed unless otherwise noticed.

Cell Culture Protocols PER.C6 cells were cultured in the examples. Cells were adapted from adherent cultures in DMEM containing 10% FBS (Invitrogen) to serum free medium by direct transfer. Briefly, sub-confluent, logarithmic cells were trypsinised, washed once 15 with serum free medium and inoculated .directly into 250 ml Ehrlenmeyer flasks with a 0.2\i filter (Corning), containing 25 ml of ExCell-525 serum-free medium (JRH Biosciences) at a starting cell concentration of 0.3-0.5 x 106 ml-1, unless otherwise noted. Cultures were maintained in logarithmic 20 growth in Ehrlenmeyer flasks by passage every 2-3 days. Flasks were shaken on a magnetic shaker platform (Infors) at 100 rpm in a humidified incubator at 37°C and 5% CO2. Cultures were passaged by centrifugation at 1000 rpm for 5 minutes. The supernatant was removed and the pellet re-suspended in the 25 remaining medium. Fresh, cold medium (4°C) was added and new flasks inoculated at the appropriate cell concentration. After transfer to serum-free medium, cultures were passaged for 2-4 weeks to allow for complete adaptation, after which a serum-free cell bank was created. All experiments were started using 30 cells from this cell bank. 18 Bioreactors Bioreactor cultures were performed in 3L reactors with a 2L working volume (Applikon). Temperature was maintained at 37°C by a heating blanket. Dissolved oxygen concentration (d02) was 5 controlled at 50% of air saturation by adjusting inlet gas composition through the headspace and intermittent sparging through a microporous sparger. Starting culture pH was controlled at 7.3 by CO2 addition through the microporous sparger. The lower culture pH limit was set at 6.7 so tha:t the 10 culture pH was allowed to drift downwards (the lower limit was not reached). Cultures were agitated by two marine impellers at 75 rpm. Process data was acquired by the BioExpert software (Applikon).

Analytical Protocols Cell counts and viability measurements were performed using a CASY automatic cell counter (Scharfe Systems). Glucose, lactate, ammonia and phosphate concentrations were determined using an Ektachem II analyser (Kodak) with cell-free culture 20 supernatants. Amino acid concentrations were determined using a modified AccuTag HPLC method (Waters) as described by van Wandelen and Cohen (1997) . Aliquots (200 |il) of centrifuged culture supernatant were stored at -20°C in 1 ml cryovials (Nalgene) until required. Samples from each experiment were 25 analyzed at the same time to avoid experimental variation. Osmolality was measured by a freezing point depression osmometer (Osmomat 030-d, Gonotec). Antibody concentration was determined by a sandwich-type ELISA. Biefly, plates were coated with 2 jig ml-1 mouse anti-human IgG against the kappa 30 light chain (Pharmingen) and incubated overnight at 4 °C. An HRP-conjugated mouse anti-human IgG against the heavy chain (Pharmingen; 1:500) was used as detection antibody for 1 hr at 19 37 °C with OPD (Sigma) as substrate. Washing between incubation steps was performed with 0.05 % Tween 20 in PBS. Samples were diluted in washing buffer supplemented with 0.1 % BSA. Quantification was relative to an IgGl reference standard 5 using a calibration range of 10 to 400 ng ml-1. Antibody samples purified by Protein A were subject to quality analysis by isoelectric focusing (IEF) and denaturing polyacrylamide gel electrophoresis (SDS-PAGE). For glycan analysis, N-linked glycans were removed by PNGase F treatment of the IgG samples 10 in 20 mM sodium phosphate (pH 7.2) and analyzed with MALDI-MS in the reflector mode on an Applied Biosystems Voyager DE Pro mass spectrometer. The matrix was 2,5-dihydroxybenzoic acid (10 mg ml-1) in 50/50/0.1 acetonitrile/water/trifluoroacetic acid. Spectra were obtained in the positive ion mode and 15 glycans were detected as sodium adducts, [M+Na]+.

Calculation of Cell Specific Metabolic Rates Cell specific rates of metabolite utilisation and production in batch and fed-batch culture were calculated using the log 20 mean of the cell concentration as shown in the following equation: qs - (C2 - Cj.) / (t2 - ti) x [<X2 - Xi) / ln(X2 - Xi)].

In this equation, C is the metabolite concentration (pmoles/1), t is time (days) and X is the viable cell 25 concentration. A rate constant accounting for the spontaneous decomposition of glutamine was not included as decomposition was not significant at the time points at which the rates were calculated (data not shown). The yield coefficients of lactate produced per glucose (Yiac/gic), ammonia produced per glutamine 30 (Yamm/gin) and alanine produced per glutamine (YaWgin) were calculated from the equations below and are expressed in mole/mole: Ylac/glc ~ Qlac / *3glc Yamra/gln ™ Cfarwr, / CJgln Yala/gLn ~ Qaia / *3gln EXAMPLES Example 1: Increasing maximum final cell yields in batch culture of PER.C6 cells The simplest production process is a batch culture. 10 However, this is restricted in the viable cell concentration and therefore the product yields attainable, due largely to nutrient limitation. A method is presented to increase the maximum final cell concentration of a batch culture of PER.C6 or PER.C6 derived sub-clones by calculating the cell specific 15 rate of utilisation of key nutrients at different cell concentrations and starting the batch culture at a cell concentration where there is optimal utilization of nutrients with respect to cell growth.

The DNA encoding the antigen-binding region of an 20 antibody recognizing epithelial cell adhesion molecule (EpCAM) was first isolated from a scFv phage display library (Huls et al, 1999). DNA encoding the antigen-binding region of an antibody recognizing CD46 was isolated as-disclosed in WO 02/018948. A leader sequence and constant regions of IgGl type 25 were added essentially as described in Boel et al, 2000. The DMA encoding the light and heavy chains were then cloned into expression.vector pcDNA3002(Neo). The expression vector pcDNA3002(Neo), which has been described in international patent application PCT/NL02/00841, was deposited on December 30 13, 2001 at the European Collection of Cell Cultures (ECACC) under number 01121318. The resulting expression vectors, encoding an IgGl that recognizes EpCAM or CD46, respectively, 21 regulated by a CMV promoter, was introduced in PER.C6 cells according to standard methods.

A recombinant antibody-expressing clone, derived from a parental population of the PER.C6 cell line, was used in these 5 experiments. The clone expressing anti-EpCAM is further referred to herein as clone 1, the clone expressing anti-CD4 6 is further referred to herein as clone 2.

Cells were maintained in ExCell™ 525 medium (JRH Biosciences) (maintenance of the cells in GTM-3 medium (Sigma) did also 10 work) and batch productions were carried out in ExCell™ VPRO medium (JRH Biosciences, Cat. No. 14560) . Cells were transferred directly from ExCell™ 525 to ExCell™ VPRO for the batch productions.

Fig. 1 shows that the maximum final viable cell 15 concentration of cultures started at 1 x 106 cells ml-1 reached almost 14 x 106 cells ml"1 after 6 days (approximately 3-fold higher than batch cultures of CHO and Sp2/0), compared to cultures started at 0.3 x 106 ml"1, which reached 10 x 10s cells ml"1 after 9 days. There is very little difference in the final 20 antibody titres of both cultures. However, in the culture started at 1 x 106 ml-1, approximately 600 mg L-1 was reached after 6 days, compared to 9 days for the cultures started at 0.3 x 106 ml"1.

The higher cell concentrations observed in cultures 25 started at 1 x 10® cells ml-1 compared to 0.3 x 10s ml"1 is due to the lower cell specific rate of nutrient utilisation at the higher cell concentration. The respiration rate of hybridoma cells has been shown to decrease with increasing cell density (Wohlpart et al 1990). Similarly, the cell specific rate of 30 utilisation of a nutrient has also been shown to decrease with increasing cell concentration (Portner et al 1994, Yallop and Svendsen 2001). We have now used this information in a novel 22 and inventive way to form a concept for increasing attainable cell densities in a culture.

By calculating the cell specific rate of utilisation of a key nutrient each day in a batch culture and plotting these 5 values against cell concentration, a graph can be obtained as shown in Fig. 2 for glutamine. Fig. 2 shows the relationship between the cell specific rate of glutamine utilisation (qGin) and cell concentration. From this graph, an optimum starting cell concentration can be selected based on optimal use of the 10 available nutrients. For example, a culture starting at 0.3 x 106 cells ml-1 will reach approximately 0.5 x 106 ml-1 in 24h (average population doubling time (pdt) of this clone is 32h). The qGin value at 0.5 x 106 cells ml-1 is approximately 2.5 [imoles 106 cells-"1 24h-1. The total glutamine consumed in this 15 24h will therefore be approximately 1.25 Jtmoles ml-1 (0.5 x 2.5) . However, a culture starting at 1 x 106 cells ml-1 will reach approximately 1.5 x 10® ml-1 in 24h. The qein value at this cell concentration is approximately 0.75 |imoles 10s cells-1 24h_1. The total glutamine consumed will therefore be 20 approximately 1.125 (Jmoles ml-1. The two cultures will therefore use approximately the same amount of glutamine in the first 24h.

It is therefore another object of the invention to provide a method of culturing cells, comprising starting a 25 culture at a cell concentration where the specific nutrient utilization level is close to a minimum plateau level. This equates with around 0.8 to 2.0 x 10® cells/ml, preferably 0.9 -1.5 x 10® cells/ml, for El-immortalized retina cells, particularly PER.C6-derived cells. It is therefore an 30 embodiment of the invention to subculture the cells at a seeding concentration of 0.8-2.0 x 106 cells/ml, preferably 23 0.9-1.5 x 106 cells/ml, more preferably 0.95-1.25 x 106 cells/ml.

The advantage of this aspect of the invention is that the number of viable cells that can be obtained is higher at this 5 higher seeding density, and higher numbers of cells are reached faster during the process. This aspect of the invention therefore is very useful for batch cultures, but can also be beneficially used in fed-batch cultures or (fed-) perfusion cultures, such as those of the present invention.

Example 2: Feed Strategies for improving antibody yields in PER.C6 derived sub-clones.

Fed-batch processes aim at increasing product yields by increasing the viable cell concentration or prolonging the 15 production period by feeding nutrient concentrates to replenish those that are consumed. We present here a feed strategy for improving the antibody yields of PER.C6 derived sub-clones. The feed strategy can be combined with a higher starting cell density to obtain a higher final cell density at 20 the onset of the nutrient feed and a shorter overall production process.

A basic nutrient feed concentrate consisting of glucose, phosphate, glutamine and the 15 other amino acids was prepared based on the nutrient utilisation profile of six duplicate 25 batch cultures of clone 1 in shake-flask (see e.g. Fig. 3). Similar utilization profiles were observed for clone 2, and hence it is expected that the feed strategy described below for clone 1 will also improve yields from other clones, thereby providing a more generic strategy for fed-batch or 30 fed-perfusion cultures of El-immortalized cells, preferably retina cells, preferably cells derived from PER.C6 cells. The concentrate is listed in Table 1. Optionally, calcium and 24 three recombinant growth factors, LongR3 IGF-1, Long EGF and insulin were also added to the feed. At this point, the addition of calcium and the growth factors did not significantly influence the results that were obtained.

Glycine appeared not essential for the feed, and was no longer added in later experiments. Insulin was purchased from Sigma, LongR3 IGF-1 and Long EGF were purchased from GroPep. All amino acids were purchased from Sigma. The timing and frequency of addition of the feed concentrates was varied. The 10 time of the first addition was tested at 0, 1 and 2 days prior to nutrient exhaustion. Glucose and phosphate were used as indicators for the start of the feed. A series of bolus additions were made every two days, based on the predicted viable cell concentration. Usually, 6 feeds were provided. The 15 concentrations of the added components as presented in Table 1 do not take into account the remaining component in the spent medium before the addition (i.e. the concentration of a component after addition into the culture medium -will be higher than that provided in the Table, because before the 20 addition the culture medium will still contain some of this component, as additions according to the invention are done before the component is completely used up by the cells) .

Fig. 4 shows the effect of feeding the concentrate mix to a sub-clone of PER.C6 expressing a recombinant antibody (clone 25 1). Starting the feed at day 3 (two days prior to nutrient exhaustion and continuing every two days after this) resulted in a final antibody yield of approximately 800 mg L"1, an increase of approximately 1.6-fold over the batch process, which gave 500 mg L"1. Starting the feed at day 5 and 30 continuing every two days after this) resulted in a similar increase in final antibody concentration.

Osmolality in the batch cultures (example 1) decreased from 280 to 240 mOsm Kg-1, while in the feed cultures, it increased, eventually rising to 300-310 mOsm Kg"1.

Example 3: Achieving viable cell numbers above 30 x 106 cells per ml and antibody yields above 500mg L"1 day-1 Fed-batch processes may result in a build-up of toxic metabolites such as lactate and ammonia and an increase in medium osmolarity, which eventually limit the viable cell 10 concentration and the length of the process, thus impacting on product yields. A possible alternative to a fed-batch process is a perfusion process, where high cell concentrations can be maintained by a continual medium exchange and a cell bleed (removing a certain percentage of the cells population). A 15 possible drawback with such a process is a relatively low product concentration due to the large volumes of medium that are required, the relatively low cell viability often encountered and the relatively high level of complexity to operate such a system. It is therefore only advantageous to 20 operate perfusion processes if very high viable cell concentrations and/or specific productivities can be maintained.

We present here the attainment of a viable cell concentration above 30 x 10s ml"1 and antibody yields of above 600 mg If1 24h-1 25 in shake flask cultures with one medium volume exchange per day.

Logarithmic cultures of antibody producing PER.C6 cells, cultured in shake flask with ExCell1" 525 were transferred into shake flasks containing ExCell™ VPRO at a starting cell number 30 1 x 10€ cells/ml (other starting cell concentrations gave similar results) .. Medium replacement by centrifugation (one volume per day) was started at day 3-5. No cell bleed was 26 operated- Samples for metabolite analysis, antibody quantification and cell counts were taken every day and stored at -20°C.

Fig. 5 shows that a viable cell number of up to 50 x 106 5 ml-1 and an antibody yield of 500-750 mg L_1 24h-1 was maintained for at least 5 days without a cell bleed, for two independent antibody producing cell clones. Viability of the cells was around 80-90%. These high cell densities are approximately 3-fold higher than is generally achievable with 10 other cell lines like CHO and Sp2/0, and hence retina cells that are immortalized with adenovirus El sequences, such as PER.C6 cells, are very suitable for perfusion processes. A cell bleed will improve the length of the process, and therefore an optimized system may include one or more cell 15 bleed steps.

Up to 50 x 106 cells per ml, with a viability of around 80-90%, could be maintained for at least 5 days with one complete medium change every two days. With this strategy, many of the nutrients became depleted on the second day. The 20 medium is therefore preferably changed daily. In a perfusion process, this could translate into a change of about 1-3 volumes/day. This is near the typical range in a standard perfusion system, where the medium is changed at about 0.5 to 2 volumes/day. The somewhat higher values for the cells 25 according to the invention are due to the very high cell concentrations with the cells of the invention in a perfusion system. When cell concentrations of more than 30 x 106 cells/ml according to the invention are preferred, the medium exchange should at least be 0.5 culture volumes/day, preferably at 30 least 1 culture volume/day. Failure to supply the nutrients (here via the culture medium) in sufficient concentration leads to cell death. The daily medium change results in higher 27 viable cell densities (up to 50 x 106 cells/ml with daily medium change vs. 10 x 106 cells/ml without daily medium change, see Figs 1 and 4). Furthermore, with a daily medium exchange, the cells give similar product yields in one day as 5 achieved in a batch process of 8-13 days.

Example 4: Feed Strategies for further improving antibody yields in PER.C6 derived sub-clones.

The provision of a balanced nutrient feed extends to 10 components such as vitamins, trace elements and lipids. Concentrates (lOx or 50x, both worked) of ExCell VPRO vitamins, inorganic salts, trace elements, growth factors, lipids and plant hydrolysates were obtained from JRH Biosciences and added together with the basic feed concentrate 15 (minus calcium and growth factors) described in example 2. The ExCell VPRO concentrates were added to give a final concentration of 0.25X.

Fig. 6 shows the results of this modified feed on the growth (Fig. 6A) and antibody yields (Fig. 6B) of clone 1 in shake-20 flask versus a batch control. The results were obtained by starting the feed at day 3 (48h prior to nutrient depletion). Starting the feed at day 5 (day of nutrient depletion) gave similar results. The viable cell number was maintained for significantly longer than the batch control and antibody 25 yields increased 2.0-fold from 0.5 g If1 in the batch to 1.0 g If1 in the fed-batch process.

Spent medium analysis of these feed experiments identified a change in the cell specific rates of utilization of some of 30 the amino acids, which appeared to be de to the addition of the VPRO concentrates. The amino acid concentrate listed in Example 2 was therefore modified as shown in Table 2. The feed 28 was started 48h prior to nutrient depletion and additions were made every two days. Usually, 6 feeds were provided. Again, the concentrations of the added components as presented in the Table do not take into account the remaining component in the 5 spent medium before the addition.

For the first feed addition, increased concentrations of Glutamine and Serine were used as compared to the subsequent feeds (see Table 2). Phosphate and glucose were used as markers to determine the start of the feed. Clones 1 and 2 10 were used in this experiment.

Experiments were carried out in shake-flask and bioreactor. Shake flask experiments were carried out as described. Bioreactor experiments were initiated by inoculating a 3L bioreactor (Applikon, 2L working volume) with cells from a 15 logarithmic pre-culture grown in shake flask. The pre-culture and bioreactor experiments were performed in ExCell VPRO (JRH Biosciences) . The split ratio for inoculation into the bioreactor was at least 1:6, and the seeding cell concentration was about 0.3 x 10® cells/ml.

Results Fig. 7 shows the results of the modified feed on clone 1 in bioreactor versus a batch control. The maximum viable cell number reached 10-12x10® ml-1 and viable cell numbers were 25 maintained between 8 and 10x106 cells ml-1 until the end of the culture at day 19 (Fig. 7A) . Antibody yields increased 3-fold from 0.4 g L"1 in the batch to 1.3 g L-1 in the fed-batch process (Fig. 7B).

Osmolality and ammonia reached 430 mOsm. Kg"1 and 16 mmoles IT1 30 respectively in these feed cultures, levels that have been reported as having negative effects on culture performance and product quality. It may therefore be that the decrease in 29 viable cell numbers observed towards the end of the process was due at least in part to these factors.

Fig. 8 shows the results of the feed strategy on clone 2 in 2L bioreactors. Maximum viable cell numbers reached 10-11 x 10s 5 ml""1 and 7-9 x 10® ml-1 were maintained until the end of the culture at 19 days. Antibody yields were increased 3-fold from 0.5 g IT1 to 1.5 g L-1.

A third clone expressing another, again unrelated, antibody was subjected to the same batch process and the fed-batch 10 process with the same feed strategy. Fig. 10 shows the results of the feed strategy on this clone 3 in shake flask. Maximum viable cell numbers reached 14 x 106 ml-1 and 10-12 x 10s ml-1 were maintained until the end of the culture on day 17. Antibody yields were increased 3-fold from 0.7 g L-1 to 2.1 g 15 I"1.

The feed strategy therefore improves the yield for different clones that each express a different antibody, indicating that the process according to the invention is generically 20 applicable.

It is therefore an aspect of the invention to provide a process comprising the feed strategy according to the invention, wherein the yield of a produced protein is increased at least 1.5 x, preferably at least 2 x, more 25 preferably at least 2.5 x, still more preferably at least 3x over the yield in the batch process.

The specific productivity (q&b) of the cells used in the present invention was approximately between 12-18 pg 30 antibody/cell/day. In some instances the q»b was around 10 pg antibody/cell/day, and in other instances values up to about 25 pg antibody/cell/day were observed with the cells and methods of the present invention. In the batch cultures this decreased significantly before maximum cell numbers were reached, coinciding with depletion of nutrients, which was approximately after 7 days, whereas in fed-batch cultures this 5 specific productivity was kept at this level until 2-3 days after the last feed addition, which amounts to around 16-18 days, according to a process of the invention.

Product Quality 10 In the experiments described above, product quality was checked by various methods, including iso-electric focusing, SDS-FAGE, MALDI-TOF mass spectrometry and HPAEC-PAD. In all cases the produced antibody basically showed a human-type glycosylation and the structural integrity of the produced 15 antibodies was very good, irrespective of the process used, and very similar to that reported in <Jones et al, 2003), where both cell numbers and product yields were lower. Therefore, the increased yields obtainable by processes of the invention were not obtained at the cost of a'significant 20 decrease in product quality.

Protein A purified IgG produced from batch and fed-batch cultures was analysed by MALDI-MS. Material produced by PER.C6 cells from batch cultures showed a galactosylation profile similar to that shown by IgG purified from human serum and no 25 hybrid or high mannose structures were identified in either batch or fed-batch produced material. The average percentage of glycans terminating in 0, 1 and 2 galactose residues (G0:G1:G2) from all the batch cultures tested was 29, 54 and 17 % respectively. This can be compared to CHO and hybridoma 30 produced antibody, which is often predominantly in the GO form. For example, Hills et al (1999) reported a 31 galactosylation profile (G0:G1:G2) for an antibody produced in NSO and CHO cells.

Antibody produced in the fed-batch process showed a reduced level of galactosylation compared to the batch (Fig. 9). The 5 percentage of GO glycoforras increased from 29 to 49%, while the G1 and G2 glycoforms decreased from 54% and 17% to 42% and 9% respectively. This decrease in galactosylation was probably due to the high (up to 16 mM) ammonia concentrations at the end of the fed-batch cultures. However, the level of 10 galactosylation in the antibody produced by the fed-batch process in PER.C6 cells was still higher than typically seen in batch-produced antibodies from CHO for example (Hills et al 1999). Isoelectric focusing (IEF) and SDS-PAGE revealed no significant differences between the material produced by batch 15 or fed-batch cultures (data not shown) and in all cases, aggregation was below 3%.

Despite relatively low Yamm/gin values, the high viable cell concentrations resulted in a supply of glutamine in the feed such that the ammonia accumulated up to 16 mmoles If1. Whilst 20 this did not result in a drop in the viable cell concentration, batch cultures initiated in the presence of NH4CI showed that concentrations above 9 nvmole If1 negatively affected growth rates and maximum cell concentrations. Furthermore, glycosylation was also somewhat affected (see 25 Fig. 9). It may therefore be beneficial to reduce ammonia accumulation, e.g. according to a method described below.

Two areas for attention in the process described so far are the high levels of ammonia and osmolality. A large contributor 30 to the increase in osmolality came from the VPRO (medium) concentrates. An approach to reduce this osmolality is therefore to identify which of the medium component groups 32 (vitamins, trace elements, inorganic salts, growth factors etc) are important to culture performance and remove those that are not important. This should benefit the process not only by reducing the osmolality of the feed but also by 5 removing any potentially deleterious components and by allowing the optimization of addition of the most important components. It would also reduce the cost of the feed. Reduction in ammonia accumulation may be achieved by more strictly controlling glutamine addition. This can be done 10 based on the calculations of the specific consumption and cell numbers as described supra. This can be achieved by continuously pumping in glutamine at an appropriate rate, matched to the viable cell concentration and the cell specific rate of utilization, so that residual glutamine concentrations 15 in the medium are maintained at a constant low level, such as between 0.2 and 1.5 mM, preferably between 0.5 and 1.0 mM. Another approach that may be possible for the cells according to the invention is the removal of glutamine from the feed when the ammonia concentration reaches a certain point -e.g. 20 in one or more of the feeds subsequent to the first feed- so that the cells are forced to switch to glutamine synthesis using ammonia and glutamate and the glutamine synthetase pathway. This approach is not generally possible for cell types such as BHK and CHO as glutamine depletion often results 25 in rapid and widespread cell death and transfer to glutamine-free conditions often requires a period of adaptation.

However, in batch cultures of the cells according to the present invention, the viable cell concentration continued to increase for two days after the depletion of glutamine and 30 culture viability was not significantly affected, suggesting that there may be sufficient flux through the glutamine synthetase pathway at least to maintain the culture- 33 Spent medium analysis of the most optimized fed-batch culture (examples in Fig. 7, 8) showed that only cystine was depleted during the process. A further modification of the amino acid feed according to the invention is therefore an increase in 5 the cystine concentration, e.g. to 0.3-0.35 mmoles/1 or even up to 0.6 mmoles/1 for every 10 x 106 cells/ml.

Example 5: improved (fed-)batch process.

Feed concentrates developed for fed-batch processes may 10 also be used to supplement culture media for use in an improved batch process. Supplementing a culture medium with at least one of the feed additions from a fed-batch process has been shown by others to improve batch yields. A similar approach of supplementing culture media with feed concentrates 15 may also be used to reduce the number of feed additions during a fed-batch process, thereby simplifying the process, as also shown by others.

The present invention discloses feed strategies for cells that have been immortalized by adenovirus El sequences, such 20 as PER.C6 cells. It is shown herein which components become limiting in a fed-batch process, and the amounts of as well as the ratio between the components that can be added to improve yields in a fed-batch process are disclosed herein. This information is used in this example to provide an improved 25 batch process. It is assumed that such a culture will contain about 10 x 106 cells/ml, as this is around the number of cells that has been observed in the batch and fed-batch cultures of the invention. In the fed-batch experiments, 6 feeds were added, with concentrations of the components as in Tables 1 or 30 2. The addition of 10%-60% of the total (i.e. the total of all 6 feeds together) feed, preferably 20%-40% of the total feed, results in an improved batch process, because the nutrients 34 will become depleted later during the culture, and hence the yields will go up because of prolonged productivity compared to the straight batch process disclosed above, where no additions are made to the culture medium. The components can 5 be added directly to the culture medium at any stage prior to depletion of nutrients from the medium, but are preferably added prior to start of the culture so that no other additions have to be made during the process (improved batch process), which makes the process very simple. Of course, this may be 10 combined with extra additions of certain components later during the process (fed-batch process), in which case less additions have to be made to make the process than in the fed-batch process disclosed above, thereby providing a simpler fed-batch process. It is therefore another embodiment of the 15 invention to provide a method for producing a product in cells immortalized by adenovirus El sequences, wherein said cells are cultured in a culture medium, characterized in that the following components are added to the culture medium in the following amounts: glucose (3.6 - 21.6 mmoles/1, preferably 20 7.2 - 14.4 mmoles/1), glutamine (6.8 - 40.9 mmoles/1, preferably 13.6 — 27.2 mmoles/1), leucine (0.40 - 2.4 mmoles/1, preferably 0.79 — 1.6 mmoles/1), serine (2.31 - 13.9 mmoles/1, preferably 4.62 — 9.24 mmoles/1), isoleucine (0.3 -1.8 mmoles/1, preferably 0.6 - 1.2 mmoles/1), arginine (0.28 -25 1.66 mmoles/1, preferably 0.55 - 1.10 mmoles/1), methionine (0.14 - 0.83 mmoles/1, preferably 0.28 - 0.55 mmoles/1), cystine (0.15 - 0.9 mmoles/1, preferably 0.3 - 0.6 mmoles/1), valine (0.27 - 1.62 mmoles/1, preferably 0.54 - 1.08 mmoles/1), lysine (0.26 - 1.58 mmoles/1, preferably 0.53- 1.06 30 mmoles/1), threonine (0.18 - 1.08 mmoles/1, preferably 0.36 -0.72 mmoles/1), asparagine (0.06 - 0.36 mmoles/1, preferably 0.12 - 0.24 mmoles/1), tyrosine (0.078 - 0.47 mmoles/1, preferably 0.16 - 0.31 mmoles/1), histidine (0.06 - 0.36 mmoles/1, preferably 0.12 - 0.24 mmoles/1), phenylalanine (0.012 - 0.072 mmoles/1, preferably 0.024 - 0.048 mmoles/1), tryptophan (0.036 - 0.22 mmoles/1, preferably 0.072 - 0.14 5 mmoles/1) and phosphate (0.45 - 2.7 mmoles/1, preferably 0.9 — 1.8 mmoles/1). The amounts between brackets are 10%-60%, preferably 20%-40%, of the amounts of 6k the feeds of Table 2. Preferably, also culture medium concentrate (lOx, 50x, or other suitable concentrates can be used) is added to an end 10 concentration of between 0.15x — 0.9x, preferably between 0.3x - 0.6x. Preferably the culture medium in these embodiments is ExCell VPRO medium. An amount of 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 single feeds (a single feed being an amount as disclosed in Table 1 or 2) is added to culture medium, and simple batch 15 processes for culturing the cells at around 10 x 10® cells/ml and producing product (e.g. antibody) according to the invention are performed with the thus fortified media, to determine the optimum amount of component additions. Improved batch processes giving the highest product yields are expected 20 when about 20%-40% of the total feed of a fed-batch process according to the invention are provided to the culture medium prior to culturing, i.e. somewhere between 1 to 2.5 single feeds. Of course, more fine-tuning of the amount is possible once a beneficial range of added components is established by 25 these experiments. Of course, when the cell numbers are different, the component addition can again be adapted. For instance, if the cells are cultured at a density of only 5 x 106 cells/ml, addition of an amount of only half the amount above would be required, as is clear to the person skilled in 30 the art. 36 Table 1 Components Glucose Glutamine Leucine 10 Serine Isoleucine Arginine Methionine Cystine 15 Valine Lysine Threonine Glycine Asparagine 20 Tyrosine Histidine Penylalanine Tryptophan Phosphate 25 Calcium LongR3 IGF-1 Long EGF Insulin Final Concentration (after addition) (per 10 x 106 cells/ml) (mmoles L-1) 6.00 1.75 0.60 0.55 0.45 0.42 0.23 0.14 0.45 0.40 0.33 0.33 0.15 0.14 0.11 0.10 0.02 0.70 0.02* 50 ug/L* 50 ug/L* ug/L* *optionally present 37 Table 2 Components Glucose Glutamine Leucine 10 Serine Isoleucine Arginine Methionine Cystine 15 Valine Lysine Threonine Asparagine Tyrosine 20 Histidine Penylalanine Tryptophan Phosphate 10X VPRO Concentrate final Concentration (after addition) (per 10 x 106 cells/nil) (mmoles L-1) First Feed Subsequent Feeds 6.00 6.00 2.60 1.75 0.66 0.66 1.10 0.55 0.50 0.50 0.46 0.46 0.23 0.23 0.25 0.23 0.45 0.45 0.44 0.44 0.30 0.30 0.10 0.10 0.13 0.13 0.10 0.10 0.02 0.02 0.06 0.06 0.75 0.75 0.25X 0.25X 38 REFERENCES Boel E, Verlaan S, Poppelier MJ, Westerdaal NA, Van Strijp JA, Logtenberg T (2000). Functional human monoclonal antibodies of all isotypes constructed from phage display 5 library-derived single-chain Fv antibody fragments. J Immunol Methods. 239, 153-66.

Hills AE, Patel AK, Boyd PN and James DC. 1999. Control of therapeutic antibody glycosylation. In: A Bernard, B Griffiths, W Noe and F Wurm (eds), Animal Cell Technology: 10 Products from Cells, Cells as Products, 255-257. Kluwer Academic Press, Dordrecht, The Netherlands.

Huls GA, Heijnen IAFM, Cuomo ME, Koningsberger JC, Wiegman L, Boel E, van der Vuurst-de Vries A-R, Loyson SAJ, Helfrich W, van Berge Henegouwen GP, van Meijer M, de Kruif J, 15 Logtenberg T. (1999). A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments. Nat Biotechnol. 17, 276-281.

Jones D, Kroos N, Anema R, Van Montfort B, Vooys A, Van Der Kraats S, Van Der Helm E, Smits S, Schouten J, Brouwer K, 20 Lagerwerf F, Van Berkel P, Opstelten D-J, Logtenberg T, Bout A (2003) High-level expression of recombinant IgG in the human cell line PER.C6. Biotechnol. Prog. 19: 163-168.

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Xie L, Pilbrough W, Metallo C, Zhong T, Pikus L, Leung J, Aunips, Zhou W (2002) Serum-free suspension cultivation of 5 PER.C6® cells and recombinant adenovirus production under different pH conditions. Biotechnol and Bioengin 80: 569-579.

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Claims (11)

WHAT WE CLAIM IS:

1. A culture of cells derived from PER.C6 cells, characterized in that said culture comprises at least 12 x 106 cells/ml.

2. A culture of cells according to claim 1, characterized in that said culture comprises at least 15 x 106 cells/ml.

3. A culture of cells according to claim 2, characterized in that said culture comprises at least 20 x 106 cells/ml.

4. A culture of cells according to claim 3, characterized in that said culture comprises at least 30 x 106 cells/ml.

5. A culture of cells according to claim 4, characterized in that said culture comprises at least 40 x 106 cells/ml.

6. A culture of cells according to any one of claims 1-5, wherein at least 80% of the cells are viable.

7. A culture of cells according to any one of claims 1-6, wherein culture medium is exchanged at a rate of about 0.2-3 culture volumes/day.

8. A culture of cells according to any one of claims 1-7, wherein cells in said culture express a recombinant protein.

9. A culture of cells according to claim 8, wherein said recombinant protein is an immunoglobulin.

10. A culture of cells according to any one of claims 1-9, wherein said culture is a suspension culture.

11. A culture of cells as claimed in claim 1 substantially as herein described with reference to any example thereof and/or the accompanying drawings. 40 intellectual property office op nz -2 m'w received